High specificity anticancer drug design process

ABSTRACT

A process for producing new anticancer drugs such that the drugs can be administered in a nontoxic, proto-drug form and, subsequent to a time delay which allows for differential concentration in the targer cancer or invasive tissues or cells, the non-toxic drug is then modified by an activation drug to selectively provide toxic levels of a pharmacologically active agent to the target issue.

BACKGROUND OF THE INVENTION

[0001] Despite advances in the discovery of antitumor agents, cancerremains a disease with very poor prognosis. Substances displaying invitro and in vivo anticancer activity are continuously reported in theliterature, but only a scant number of those substances go past a phaseII clinical trial. Moreover, the cure rates of drugs for the treatmentof cancers are unacceptably low, and the side effects of these drugs aresevere. The toxic effects of the drugs on the cancer tissues onlymarginally exceed their toxic effects on normal, healthy cells of thebody that should be protected from the effects of the drugs. Moreover,that high failure rate of potential anticancer agents arguably can betraced to the lack of methods to ascertain and to design into, inadvance, control of secondary effects, specificity, and hightarget-tissue activity in human subjects. A design process is needed todesign into candidate compounds all the requirements for the drugdelivery system and anticancer effectiveness. That goal is achieved bythe present invention.

[0002] Chemotherapy research in cancer treatment has been largelydevoted to the search for drugs providing toxin specificity to destroyneoplastic tissue in the body without exceeding toxic exposure levelsinjurious to healthy tissues, that is to say, to the search forcytotoxic drugs that concentrate in neoplastic tissues, or for drugsthat metabolize into such toxins. Although major efforts to this endhave been made, success has been achieved for only a few cancer types.For most cancer types, success has been quite limited.

[0003] Modem anticancer drug development began with the recognition thatsome poison gas exposures in WWI actually had anticancer side effects.The process of anticancer drug development began at this point with themostly random procedure of searching through known toxins to determinethrough exposure if the given toxin had differential toxicity that wouldaid in the control or cure of cancer. The approach centered on thediscovery of “single moiety” compounds.

[0004] In the 1930s, more complex compounds were developed so designedas to divide the function of the anticancer agent into two parts, or twomoieties on the same compound, wherein one moiety would serve toconcentrate the drug in the cancer cell or tissue and the second moietywould serve to destroy the cancer cells wherein the compoundconcentrated. A fundamental advance in this area was the elucidation ofchemical entities with tumor tissue selectivity. Early reports on suchselectivity were made for the dye Nile blue by Lewis, et al. in CancerRes. 9:736, 1949, a report that prompted the modification of thisbenzophenoxazine to incorporate a toxin such as a nitrogen mustard as anintegral part of the molecule (Sen, et al. in Int. Union against CancerActa, 26, 774 (1960)). Those attempts at modification of a tumor tissueselective agent with a toxic moiety met with limited success. It hasbeen presumed that the failure of such approaches is due to intrinsicmodification of the tissue selectivity of the parent compound with theaddition of the toxic moiety. The cause of the failure, however, is morefundamental and is shared by other drug delivery concepts, inparticular, the concept of anticancer prodrugs.

[0005] Beginning in the early 1950s, the two-moiety drug design approachof a concentrating moiety and a toxic moiety was modified to thetwo-moiety “prodrug” design, wherein one moiety served to modify or“mask” or “cap” the toxicity of the second toxic moiety of the drugcompound until the drug entered the cancer cells. At this point, achemical reaction, generally intended to be with enzymes specific to thecancer cells, would remove the mask or cap moiety so that the toxicmoiety would kill the cancer cells. Again, the prodrug anticancerdelivery concept has had limited success.

[0006] Both the concept of drugs that(combine a concentrating moietytogether with a toxic moiety and the concept of prodrugs that are to bemetabolized preferentially in cancer tissues suffer from the problemsthat arise out of the nature of cancer itself: cancer cells and tissuesare not foreign biological organisms having a chemistry distinct fromnormal tissue. These tissues are, in the case of human cancers, humantissues. The normal and neoplastic tissue have essentially the samechemistry; that is, for the most part, both consist of the same chemicalmolecules and in similar concentrations.

[0007] In 1980, Evan Harris Walker in Perspectives in Biology andMedicine, Spring Issue, 424-438, (1980) argued that the prodrug conceptand the combination of toxin with concentrator concept fail because ofthe fundamentals of the pharmacokinetics of drug delivery. The timecourse of the drugs depends on their metabolic uptake and eliminationrates. If the rates are lower for the cancer cells than for the normalcells, the drugs will appear to concentrate. That is to say, after asufficient lapse of time, the drugs will have been metabolized andeliminated out of the normal tissues while still being in significantconcentrations in the cancer cells and tissues. This will be the caseeven though the total amount of drug delivered to both normal and cancercells may be essentially the same. The problem of achieving an effectiveanticancer drug was not one of achieving differential concentration, orof having the drug be activated by enzymes in the cells, but rather oneof being able to suppress the toxicity of the drug in the normal cellsand tissues until the differential concentration of the drug had becomefavorable.

[0008] In order to take advantage of the drug concentration differentialbetween normal and cancer tissue, Walker in Perspectives in Biology andMedicine, Spring Issue, 424-438, (1980) proposed a design procedureconsisting of two drugs, a prodrug and an “activation” drug. Both theprodrug (that had a design limited to a toxin plus a cap or mask moiety)and the activation drug were to be designed so as to have little or notoxic or other effect on the body (i.e., to be substantiallybiologically inert) if administered alone. However, in combination,these two compounds would react in the body to produce a compound toxicto the cells wherein it was produced. These two drugs were to beadministered sequentially with a time delay between their introductionto allow for the normal cells to metabolize and eliminate the prodrugout of the body before the activation drug was administered. At thatpoint in time—after a time delay for differential delivery of thedrug—differential concentration of the prodrug would have been achieved.Removing the masking or capping moiety of the prodrug at that time—whenthe prodrug would be differentially concentrated in the cancertissues—would then result in delivery of high levels of the includedanticancer toxin. In this way, anticancer toxins would be delivered tocancer cells and tissues without harm being done to normal cells andtissues.

[0009] Prodrugs of the type proposed by Walker were designed andsynthesized by the present inventors. One of these was a5-fluorouracil-N-glucoside, which can be activated by the enzymeβ-glucosidase. After designing a prodrug of this type, the inventors ofthe present disclosure found that a more complex prodrug and activationdrug system would be needed to provide all the requirements for aselective and effective anticancer pharmaceutical. In particular, thepresent inventors found that the prodrug and activation design of thistype also had limitations in that the differential concentrationachievable within the limits of that design was not sufficiently high.In other words, the differential concentration effect due entirely todifferential diffusion with no added moiety to enhance the concentrationdifferential proved to be inadequate. A more complex drug system wasneeded and it was found that a process for the development of the drugsystem would be required.

SUMMARY OF THE INVENTION

[0010] The present disclosure covers an assemblage containing chemicalsand chemical moieties, a process for the detailed design of anassemblage, products or devices needed to carry out and evaluate theprocess, chemical systems designed by the process that serve as specificassemblage examples, and procedures needed for the successful use of anassemblage and the specific examples of assemblages designed by theprocess disclosed herein.

[0011] The assemblage covered by the present disclosure is hereindefined as a complex proto-drug and activation drug system. Theproto-drug includes one or more differentially selective moieties, oneor more toxic moieties, and one or more moieties that serve the purposeof providing a “mask” or “cap” to the toxic moieties.

[0012] The present disclosure also covers an activation drug, hereindefined as one or more chemicals serving the function of activating theproto-drug of the assemblage. The activation drug removes the mask orcap moiety, or a sufficiency of the mask or cap, or so modifies thechemistry of the overall proto-drug molecule as to substantiallyeliminate the masking or capping function of the mask or cap, therebymaking the resulting compound a toxic, pharmacologically active agent.In addition, the present disclosure covers the introduction of “links”that serve the purpose of chemically tying the moieties of theproto-drug together so as to form a single compound, that alone issubstantially biologically inert. The links may be the chemical bondsbetween the linked moieties or a combination of atom(s) and bonds thatconnect the moieties of the proto-drug.

[0013] The present disclosure also covers a process whereby theproto-drug and activation drug system (i.e., the assemblage) is to bedesigned and specified. That is to say, the likelihood that such acomplex system of chemicals having the set of required properties couldbe obtained through chance experimentation alone is nil. As a result, itis necessary to develop a process whereby such chemical systems can beproduced.

[0014] The present disclosure also covers novel products or devices usedin the present process.

[0015] The present disclosure also covers specific examples ofproto-drug and activation drug systems (i.e., assemblages) as producedby the successful use of the process.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The assemblage, the process for the detailed design of anassemblage, products or devices needed to carry out and evaluate theprocess, and procedures needed for the successful use of the examples ofthe chemical system presently disclosed all serve to provide for ahighly selective and effective anticancer pharmaceutical. The examplesand corresponding data set forth below serve as specific assemblageexamples and demonstrate the successful use of the disclosed process.The process is not limited to the design of an anticancer agent but mayalso be used to develop other pharmaceuticals where toxicity in a targetcell population is required with concomitant low toxic side effects.

[0017] An object of the present invention is to provide an assemblage,which assemblage includes a proto-drug and an activation drug, wherebythe proto-drug is made of at least one differentially concentratingmoiety, at least one toxic moiety and at least one cap moiety. Thedetails of the components of the proto-drug are:

[0018] 1. The differentially selective moiety or moieties (also referredto as the “differentially concentrating moiety”) of a chemical compoundhaving properties such that the compound will differentially concentratein cancer tissues as compared with normal tissues of the treated bodywhether in animals or people. Here differentially concentrate means thatthe ratio of the concentration of the said compound in the cancer tissueas compared to the concentration of that compound in normal tissues willat some time during the pharmacokinetics of the drug become elevated asa result of the difference between absorption, distribution, metabolism,and elimination processes of the drug in. the cancer cells as comparedto normal cells.

[0019] 2. The toxic moiety or moieties of a chemical compound havingproperties such that the compound will be capable of killing the cellsor tissues wherein it is concentrated.

[0020] 3. The mask or cap moiety or moieties of a chemical compoundhaving properties such that the proto-drug's toxicity will not beexpressed, that is to say, not act as a toxin in the cell or in thetissue where it is located, until the proto-drug is activated by theapplication or administration of the activation drug. The term mask orcap moiety is herein defined to be any modification in the proto-drugserving to mollify or eliminate the toxicity of the overall compoundwhereby such cap is later removed or modified by the activation drug.The term mask or cap need not be, and is not limited to being, achemical moiety that literally covers, or is chemically bonded directlyto the toxic moiety or to the toxic site of the proto-drug.

[0021] 4. The linkages, meaning chemical bonds or a combination of atomsand bonds between elements of the separate moieties of the proto-drugthat include at least one of each of the differentially selectivemoiety, the toxic moiety, and the cap moiety. Examples of linkages madeup of a combination of atoms and bonds include but are not limited tosingle atoms such as oxygen that can provide a linkage —O— with two bondsites to join two of the required moieties, or groups of different atomssuch as an amine (>N—CH₂—) that can link three moieties, or an azine(>C:NN:C<) that can link four moieties, if need be, together into asingle molecule.

[0022] The activation drug of the assemblage is one or more chemicalcompounds separate from the proto-drug. The activation drug serves thepurpose of activating the proto-drug by physical or chemicalmodification so as to render the resulting compound a toxic,pharmacologically active agent or so as to cause the proto-drug torelease a toxic moiety. Both the proto-drug and the activation drug areto be designed so as to have little or no toxic or other effect on thebody (i.e., to be substantially biologically inert) if administeredalone. However, in combination, the proto-drug and the activation drugwill react in the body to produce a compound toxic to the cells whereinsuch compound is produced.

[0023] The fact that the proto-drug and the activation drug are designedso as to have little or no toxic effect on the body when administeredalone uniquely distinguishes the disclosed drug design process frompreviously disclosed methods of drug development. It moves specificelements of the chemical design out of the area of biochemistry and intothe predictable discipline of organic chemistry. What has beenaccomplished by the instant invention is the modification of aconventional prodrug design. Conventional prodrugs depend on thespecifics of biochemical reactions within body tissues to be convertedinto a pharmacologically active compound. The proto-drug of the instantinvention is a substantially biologically inert compound that isamenable to reaction with, for example, an inorganic compound such thatalthough the reaction takes place within the environment of a biologicalsystem, it is not susceptible to reaction with the endogeneous moleculesof the biological system itself. Once the proto-drug and the activationdrug react, the products of that reaction and their metabolites becomecompounds having well known biological behaviors that retain theirproperties in well explored and well studied fashion and under theaddition of many chemical moieties. What will happen to these chemicalsthat go into making up the proto-drug is predictable over a wide rangeof chemical modifications. Mechlorethamine, as a specific example,remains a toxin to the biological tissues in which it is deposited for alarge number of modifications—modifications well studied and understood.Thus, both the proto-drug precursor chemistries that are “a-biologic”and the chemistries of their corresponding pharmacologically activereaction products are sufficiently understood as to allow fordetermination of modifications that are the basis for any number ofspecific variations in the instant specification and that are amenableto expansion within known teaching.

[0024] The process for the detailed design of an assemblage includes thefollowing process steps:

[0025] Selection of a Differentially Concentrating Moiety: A processstep whereby chemical moieties of a compound having properties enhancingdifferential concentration, meaning that the ratio of the concentrationof the said compound in the cancer or other targeted tissue or tissuesas compared to the concentration of the compound in normal tissues willat some time during the pharmacokinetics of the drug as a result ofabsorption, distribution, metabolism, and elimination processes, becomeelevated, are identified by specific procedures. One method of selectionof compounds containing a differentially selective moiety is HPLC. TheseHPLC procedures include, but are not limited to, subjecting candidatecompounds containing (or consisting entirely of) the differentiallyconcentrating moiety to a determination of diffusion rates throughvarious types of HPLC columns to select for those having low diffusionrates (that is, high retardation rates). Examples of HPLC columns usefulfor this purpose are cancer cell RNA doped HPLC columns, cancer cell DNAdoped HPLC columns and HPLC columns doped with cancer cells, cancer cellpuree or cancer cell extract (otherwise referred to as “cancer-typecolumns”). The differentially concentrating moiety is also evaluated bya reference column, meaning an HPLC column doped with RNA or DNA fromnormal cells or doped with normal cells, normal cell puree or normalcell extract. A compound containing a differentially concentratingmoiety is selected by comparing the diffusion rate of the compound onthe HPLC cancer-type column to the diffusion rate on the referencecolumn. Those compounds having low diffusion rates (i.e., highretardation rates on the cancer-type columns) are potentialdifferentially selective moieties.

[0026] A second method of selection of compounds containing adifferentially selective moiety involves chromatography techniques otherthan BPLC. These procedures include, but are not limited to subjectingcandidate compounds containing (or consisting entirely of) thedifferentially concentrating moiety to a determination of diffusionrates through cancer cell RNA and or DNA doped chromatography columns,sheets, layers, surfaces or other configurations used in chromatographyto select for those having low diffusion rates (that is, highretardation rates), relative to the diffusion rates determined throughthe reference chromatographic system. The candidate compounds containing(or consisting entirely of) the moiety may also be subjected to adetermination of diffusion rates through cancer cell, cancer cell puree,or cancer cell extract doped chromatography columns, sheets, layers,surfaces or other configurations used in chromatography. As with theHPLC procedure, a differentially concentrating compound is selected bycomparing the diffusion rate of the compound on the cancer doped systemto the diffusion rate on the reference system.

[0027] A third method by which compounds containing a differentiallyselective moiety are chosen is by in vivo methodology. Introduction ofthe material into animal models having induced cancer growths andexamination of biopsies taken at various time delays, or sacrifice ofthe animals at successive time delays so as to make possible adetermination of the candidate material's concentration as a function oftime in both the cancer tissues and in normal tissues are in vivomethods used to evaluate potential compounds with a differentiallyconcentrating moiety.

[0028] A mechanism by which potential compounds containingdifferentially selective moieties can be chosen to be put through theabove described screening methodologies involves selection of suchcompounds from lists of dyes having high staining properties used incancer diagnostics. In this regard, it is to be noted that dyes are usedto stain biopsy tissue samples taken from patients for the purpose ofdetermining the presence of cancer, since the cancer cells have a highuptake of the particular dyes used for this purpose. Examples of suchdyes are: hematoxylin and eosin stains used in sentinel lymph nodemapping methods of evaluation in breast tissue biopsies of cancerpatients, and cytokeratin used in immunohistochemical staining of biopsytissue taken from the lymph nodes to diagnose micrometastatic disease(Pendas, et al., Annals of Surgical Oncology 7(1), 15-20, 2000,January-February). Other dyes having tissue selectivity that translatesinto tumor selectivity include Nile blue (Lewis, et al. Cancer Res. 9,736, 1949). In addition, we have noted that the yellow thioxanthonesdisplay tissue selectivity (Miller, et al. U.S. Pat. No 5,346,917). Theselectivity of these dyes, coupled with their favorable physicochemicalproperties, makes them good candidates for the selective delivery ofdrugs.

[0029] A list of such dye materials that are compounds containing, orconsisting entirely of potential differentially selective moietiesincludes, but is not limited to the following: Nile blue; thioxanthones;hematoxylin stain; eosin stain; cytokeratin stain; DNA/RNA stainsincluding trypan-blue, methyl-green, ethidium bromide, leucofuchsin dye,methylene blue; materials in references as Handbook of FluorescentProbes and Research Chemicals [by Richard P. Haugland, Sixth Edition]including the stains Acridine homodimer, acridine orange, actinomycin D,7-aminoactinomycin D, 9-Amino-6-chloro-2-methoxy-acridine,4,6-diamidino-2-phenylindole, dihydroethidium,4′,6-(diimidazolin-2-yl)-2-phenylindole, ethidium-acridine heterodimer,ethidium diazide, ethidium homodimer-1 and -2, ethidium monoazide,hydroxystilbemidine, methanesulfonate; general purpose biological stainssuch as safranin, malachite green, eosin yellowish, crystal violet,methylene blue, hematoxylin, bismarck brown, carmine alum, methyl green,and neutral red, Giemsa stain, and Gram stain, and chemicals known to‘adhere to’ but not bind to DNA and RNA sometimes referred to asimpermanent stains.

[0030] The list of potential dyes and biological stains for thedifferentially selective moiety is long. Choosing preferred candidatesfor a chemical moiety having properties enhancing differentialconcentration must be confirmed by one or more of the previouslydescribed selection methods.

[0031] Of particular concern is that the selected material be of lowtoxicity to normal tissue and to the extent that it manifests anytoxicity, that the toxicity be differentially selective against thecancer tissues (otherwise the said characteristic would compete againstthe overall drug design). This particular concern aided in the selectionof the differential concentration moiety of the proto-drug of Formulas Iand II, given below.

[0032] Selection of a Toxic Moiety: A process step whereby a chemicalmoiety having properties of causing or enhancing cytotoxicity or celldeath is determined by in vitro tests, in vivo tests, or is selectedfrom existing lists of compounds known to have cytotoxic properties andthat contain a cytotoxic moiety.

[0033] Selection of a Cap Moiety: A process step whereby chemicalmoieties having the property of masking, capping, mollifying oreliminating the cytotoxicity of the toxic moiety together with theproperty that such a cap moiety is not chemically removed by enzymaticor other metabolic processes in the body of the patient, are determinedby in vitro tests, or are selected from existing lists of reagents withthe toxic moiety selected as described above. The adequacy of the capmoiety is ascertained subsequent to the formation of the completeproto-drug (including the selected linkages as discussed herein below).More specifically, the proto-drug is tested for adequacy in vitro or inanimal models, or in human subjects. The metabolic eliminates,by-products, or extracts from the test system are evaluated by HPLC orother analytical methods to confirm that the proto-drug has not beenmetabolized or modified in such a way as to yield a toxin when appliedor administered alone (i.e., in the absence of the activation drug).

[0034] Selection of an Activation Drug: A process step whereby one ormore chemical compounds separate from the proto-drug having the propertyof removing the mask or cap moiety, or a sufficiency of the mask or cap,or so modifying the chemistry of the overall proto-drug molecule as tosubstantially eliminate the masking or capping function of the mask orcap thereby making the residual compound toxic, or so as to cause theproto-drug to release a toxic moiety within the body of the cancerpatient and in the environs of the cancer cells.

[0035] Selection of the Linkages of the Proto-Drug: The selectedchemical moiety or moieties having properties enhancing differentialconcentration, the selected chemical moiety or moieties having toxicproperties, and the selected chemical moiety or moieties having theproperty of capping the cytotoxicity of the overall chemical compound(i.e., the proto-drug) must be linked by either chemical bonds or acombination of chemical bonds and atoms between the several moietiesthat will chemically hold the moieties together allowing therein eachmoiety to facilitate the individual functions for which each moiety wasselected. These connections between the moieties are referred to as thelinkages. Generally, the method of linkage of each pair of moieties willbe chosen according to existing teaching regarding methods of bonding soas to avoid the modification of the individual properties of themoieties selected e.g., recognizing therein that the cap moiety servesto inhibit the activity of the toxic moiety until the addition of theactivation drug. In general, for each compound selected to become (afterbonding or linking) a moiety of the new proto-drug, one determines thesites responsible for the desired properties of the selected compound;it is important that these sites not be substantially disturbed ormodified by the linkages. Modifying the molecule at the severalavailable bonding positions allows one to test and determine the sitesresponsible for the desired properties. The individual moleculesselected to provide differentially concentrating and toxic propertiesfor the proto-drug are then attached at bonding positions situated awayfrom the sites active for the particular trait for which the moiety hasbeen selected; the individual molecule providing the cap moiety is thenattached at a position so situated as to inhibit the activity of thetoxic moiety. This procedure has been carried out in the process of thedisclosure, resulting in the examples of the present invention.

[0036] As a result of the process of the disclosure used to design theassemblage, the present invention provides proto-drugs of Formula I andII

[0037] wherein:

[0038] R¹ is SiZ₃;

[0039] R² is methyl, chloroethyl, hydroxyethyl, or bromoethyl;

[0040] R³ is chloroethyl, hydroxyethyl, or bromomethyl;

[0041] R⁴ is H, SO₃H, or taurine;

[0042] R⁵ is SiZ₃;

[0043] R⁶ is H, SO₃H, or taurine;

[0044] each Z of Z₃ is independently t-butyl or methyl;

[0045] X is carbon, oxygen, or nitrogen; and

[0046] W is carbon, oxygen, or nitrogen.

[0047] The present invention further provides a method of selectivelydelivering a cytotoxic compound to tumor tissue by the use of aproto-drug having a moiety that differentially concentrates, a cytotoxicmoiety and a cap moiety, whereby the proto-drug delivers the toxicmoiety in such a manner as to prevent significant damage to normaltissues by maintaining the cap moiety on the proto-drug until theproto-drug differentially concentrates in the tumor tissue during a timedelay period, and after such time delay the proto-drug produces acytotoxic compound upon administration of an activation drug.

[0048] Another object of the present invention is to provide a method ofselectively delivering a cytotoxic compound to tumor tissue byadministering a proto-drug of Formula I or II

[0049] whereby:

[0050] R¹ is SiZ₃;

[0051] R² is methyl, chloroethyl, hydroxyethyl, or bromoethyl;

[0052] R³ is chloroethyl, hydroxyethyl, or bromomethyl;

[0053] R⁴ is H, SO₃H, or taurine;

[0054] R⁵ is SiZ₃;

[0055] R⁶ is H, SO₃H, or taurine;

[0056] each Z of Z₃ is independently t-butyl or methyl;

[0057] X is carbon, oxygen, or nitrogen; and

[0058] W is carbon, oxygen, or nitrogen

[0059] so that the proto-drugs of Formulas I and II deliver the toxicmoiety in such a manner as to prevent significant damage to normaltissues by maintaining the proto-drug's cap moiety intact until theproto-drug differentially concentrates in the tumor tissue during a timedelay, and after such time delay the proto-drug produces a cytotoxiccompound upon administration of a fluoride salt.

[0060] Yet another object of the disclosure provides compounds ofFormulas III and IV

[0061] wherein:

[0062] R¹ is H;

[0063] R² is methyl, chloroethyl, hydroxyethyl, or bromoethyl;

[0064] R³ is chloroethyl, hydroxyethyl, or bromomethyl;

[0065] R⁴ is H, SO₃H, or taurine;

[0066] R⁵ is H;

[0067] R⁶ is H, SO₃H, or taurine;

[0068] X is carbon, oxygen, or nitrogen; and

[0069] W is carbon, oxygen, or nitrogen

[0070] or a pharmaceutically acceptable base addition salt of FormulasIII and IV.

[0071] This invention also provides a method of evaluating anticanceractivity for compounds of Formulas I and II with (or in the case ofFormulas III and IV, without) the activation compound, such method to beused in concert with the process for the detailed design for anassemblage and used in the process steps as detailed above, or for thein vitro and in vivo evaluation of the anticancer activity of any otherchemical substance.

[0072] This invention also provides the use of a compound of Formula Ior II with, or in the case of Formulas III and IV without the activationcompound, for the manufacture of a medicament for the treatment ofneoplasms. Additionally, this invention provides a pharmaceuticalpreparation for the treatment of neoplasms comprising an effectiveamount of a compound of the Formula I or II together withpharmaceutically acceptable excipients, and an activating amount of afluoride salt together with pharmaceutically acceptable excipients,whereby the compound of Formula I or II and the activation drug arepackaged for individual administration. Furthermore, this inventionincludes a method for the treatment of neoplasms comprisingadministering an effective amount of compounds of the Formula I or II,waiting for a time delay period and administering an activating amountof a fluoride salt.

[0073] The compounds of the invention, namely the proto-drug and theactivation drug, can be administered orally, intraperitoneally (ip),intravenously (iv), percutaneously, intramuscularly, intranasally, orintrarectally. The route of administration will vary depending on theparticular drug being administered, the disease being treated, theconvenience of the patient and the caregiver, and other relevantcircumstances.

[0074] The pharmaceutical compositions of the invention are prepared byprocedures well known in the pharmaceutical art. The carrier orexcipient may be solid, semi-solid, or liquid material that can serve asa vehicle or medium for the active ingredient. Suitable carriers orexcipients are well known in the art. The pharmaceutical composition maybe adapted for oral, inhalation, parenteral, or topical use and may beadministered to the patient in the form of tablets, capsules, aerosols,inhalants, suppositories, solutions, suspensions, or the like. Preferredcompositions and preparations of the present invention may be determinedby methods well known to the skilled artisan.

[0075] The compounds of the present invention may be administeredorally, for example, with an inert diluent in a capsule or compressedtablet dosage form. For the purpose of oral therapeutic administration,the compounds may be incorporated with excipients and used in the formof tablets, pills, troches, capsules, elixirs, suspensions, syrups,wafers, chewing gums and the like. The active ingredient in thesepreparations can vary in concentration from as little as about 0.001% toabout 70% of the weight of the unit. The amount of the compound presentin compositions is such that a suitable dosage will be obtained. Thedose range of the proto-drug and activation drug, based on customarypractice of projecting from animal models for the treatment of humans isabout 1 μg/kg to about 25 mg/kg.

[0076] On a one-to-one molarity basis, the activation drug dosage can bereduced by a factor of 10. The higher than one-to-one molarity basis forthe activation drug is given to enhance the efficacy and speed of theactivation process. In the case of children, elderly or theexceptionally infirm, dosages of the proto-drug and of the activationdrug as much as ten times lower than would routinely be used may berequired. Because experiments have shown that the unactivated proto-drugis of exceptionally low toxicity, more aggressive cancers may requiredosages of the proto-drug and of the activation drug as much as tentimes higher than for less aggressive disease.

[0077] The tablets, pills, capsules, troches, and the like may alsocontain one or more of the following adjuvants: binders such aspovidone, hydroxypropyl cellulose, microcrystalline cellulose, orgelatin; excipients or diluents such as starch, lactose, or dicalciumphosphate; disintegrating agents such as sodium starch glycolate and thelike; lubricants such as magnesium stearate, stearic acid, or talc;wetting agents such as sodium lauryl sulfate or polysorbates; sweeteningand flavoring agents such as sucrose, aspartame, peppermint and othernatural and synthetic flavors. When in capsule form the dosage maycontain a liquid carrier such as fatty oil. Tablets may be coated withsugar and binders or materials such as polymethacrylates, or othercoating agents.

[0078] Time Delay Toxin Activation and the Time Delay Period

[0079] Toxicity arises from chemical reactions that destroy compoundsvital to normal cell functions or from the production of compounds thatdisrupt cell metabolic processes. The cumulative toxic damage producedin the cell at any time is proportional to the exposure of the cell tothe toxins, that is, to the toxic exposure. Therefore, to achieveselective toxicity in the treatment of cancer, it is vital to considerthe differences in the metabolic rates that exist between normal tissuesand malignant neoplastic tissues, as, for example, has been done in thework of Chello et al., Cancer Res., 37:4297, 1977. Furthermore, thedifference between the metabolism of compounds by neoplastic and normalcells has led to the development of chemical tests for the presence ofcancer (Anderson, et. al., Cancer Res., 30, 1344 (1970); Dewanjee, et.al., J. Nucl. Med., 14, 624 (1973); Henderson, et. al., Cancer Res., 25,1018 (1965); Holland and Sleamaker, Acta Cytol., 13, 246 (1969); Máleket. al., Cas. Lek. Cesk., 1, 16 (1963); Philips, et. al., Am. J Surg.,100, 384 (1960); and Rall et. al., J. Natl. Cancer Inst., 19, 79(1957)).

[0080] The timing of the administration of the proto-drug and theactivation drug is critical if the proto-drug concentration differentialbetween the cancer cells and the normal cells is to be advantageouslyused to produce a nontoxic, effective treatment. Chemically triggeredtime delay toxin activation (“TDTA”), originally proposed by E. H.Walker in 1980, envisioned two compounds to be administeredseparately—with a time delay between the administration of the prodrug,the first drug in the sequence, and the administration of the activationdrug, the second in the sequence. In that proposal the prodrug waslimited to two moieties, the toxic moiety and the mask or cap moiety. Inthe current invention the proto-drug requires at least three parts: thedifferentially concentrating moiety, the toxic moiety, and the mask orcap moiety. The differentially concentrating moiety is an essentialdelivery moiety of the proto-drug, providing the complete molecule witha cytometabolic rate differential that with time increases theconcentration of the proto-drug in cancer cells and tissues as comparedto the proto-drug concentration in normal cells and tissues of the body.The second drug administered, the activation drug, activates theinitially administered proto-drug by removing or modifying the action ofthe cap moiety of the proto-drug. Individually each of these two drugsis designed to be substantially non-toxic (i.e., substantiallybiologically inert) when administered alone in dosage strengths.Combined, however, these two react to form or to release an anticancercytotoxin (i.e., a pharmacologically active agent). The TDTA mechanismtakes advantage of the neoplastic to normal tissue proto-drugconcentration ratio rise. with time that can occur as a result of thepharmacokinetics of agents having differing and favorable metabolicabsorption and elimination rate coefficients, as discussed below. Highuptake and low elimination rates in the neoplastic tissue relative tothese rates in normal tissues favors the long-term rise in theconcentration ratio. By measuring the kinetics of the process, as hasbeen done to establish dosages and time delays in the application of theexamples of the present disclosure, toxicity can be avoided in thenormal tissues and efficacy in the treatment of the cancerous tissuescan be effected. The mechanism involved in TDTA is fundamentallydistinct from prior chemotherapeutic approaches to toxin specificity,including those approaches involving prodrugs, latent activation drugs,and chemically targeted or site-binding drugs. While binding of thedifferentially concentrating moiety to a cell site as an aid to deliverycan be employed, the present proto-drug design is not so limited as torequire such binding to create a concentration differential and istherefore more flexible as a method of drug delivery design.

[0081] In the pharmacokinetics of normal and neoplastic tissues, uptakeand elimination of metabolic products are governed by the chemicalkinetics of cells and the physical processes of diffusion, membranepermeability, active transport, blood circulation, and excretion. To aconsiderable extent, drug concentrations can be predicted fromfirst-order kinetics equations (see below). The loss of a drug fromserum (the drug reservoir) following intravenous injection, for example,follows chemical kinetics in which uptake by body tissues takes place ona molecule-by-molecule basis (i.e., in essence the drug molecules do notreact with each other). This scenario is described by equation (1),which indicates that rate of loss from the reservoir is proportional toconcentration:

dD/dt=−pD  (1)

[0082] where D is the drug concentration (e.g., the proto-drugconcentration) in the reservoir, t is the time, and p is aproportionality constant. Equation (2) demonstrates an exponentialdependence of the drug concentration in the reservoir that is availableto the tissues at any moment:

D=D _(o) e ^(−pt)  (2)

[0083] where D_(o) is the initial serum drug concentration (at themoment of injection). The concentration C of the drug in the tissue willdepend on the drug supply in the reservoir (leading to its increase bydiffusion into the cells) and the rate at which such drug is removedfrom the tissue. This gives the rate equation:

dC/dt=gD−bC  (3)

[0084] where g and b are rate coefficients. If the concentration of thedrug in the tissue is initially zero, equation (3) has the solutionrepresented by equation (4):

C=k(1−e ^(−at))e ^(−bt)  (4)

[0085] where k=gD_(o)/a and a=p−b. The concentration-versus-timedependence given by equation (4) is encountered in tests of uptake andremoval of nutrients and drugs in tissues. Data demonstrating aconcentration-versus-time pattern for several compounds and differingmurine tissues was reported in Cancer Research, 15:365, 1955 by H. Bush.The present inventors have also carried out similar experiments withcompounds containing the thioxanthone moiety showing this same behaviorin various murine tissues over time.

[0086] Consider the circumstance where a>>b, that is, when uptake israpid. The exposure of a cell to the presence of the drug, representedby E, would then be given by the integral of C, specifically

E=∫ _(o) ^(∞) C dt=k∫ _(o) ^(∞) e ^(−bt) dt=k/b  (5)

[0087] For two cell types having elimination constants b_(A) and b_(B),the exposure ratio would be

R=E _(A) /E _(B) =b _(B) /b _(A)  (6)

[0088] This means that the exposure to a slow acting toxin would only bein proportion to the ratio of the diffusion constants for the two celltypes. Because, however, the chemistries of cancer and normal cells aresimilar, the expected exposure ratio will usually be modest—resulting inthe failure to achieve differential toxicities in the treatment ofcancers.

[0089] If the toxin of the proto-drug is masked for a time delay periodT (also referred to as “time delay”), equation (5) becomes

E=∫ _(T) ^(∞) C dt=k∫ _(T) ^(∞) e ^(−bt) dt=ke ^(−bT) /b  (7)

[0090] where E is now a function of the time delay T. In such a case,the exposure ratio becomes

R=E _(A) /E _(B)=(b _(B) /b _(A)) exp[(b _(B) −b _(A))T]  (8)

[0091] Equation (8) shows that if the elimination rate for cell type Bis larger than that for cell type A, the exposure ratio R can be made aslarge as desired if the time delay for activation is also made largeenough.

[0092] Equation (8) represents the essence of the TDTA strategy, wherebythe exposure ratio given by equation (8) can significantly exceed thatratio given by equation (6). The usefulness and validity of this novelapproach to the design and development of cancer formulas,pharmaceuticals, and application protocols has been confirmed throughthe testing of proto-drugs, activation drugs, and assemblages in thisdisclosure.

[0093] In order to convert the substantially biologically inactiveproto-drug to a pharmacologically active compound, however, anactivation drug must be administered. As with the proto-drug, theconcentration of the activation drug will be affected by the loss of theactivation drug from the serum reservoir following its administration;this process can be represented by an equation of the same form asequation (1), which demonstrates that the rate of loss from thereservoir is proportional to concentration:

dA/dt=−qA  (9)

[0094] where A is the activation drug concentration in the reservoirafter the injection time t, where t>T, and q is the proportionalityconstant for the activation drug (corresponding to p in equation (1) forthe proto-drug). Equation (10) yields an exponential dependence of thedrug concentration in the reservoir available to the tissues at anymoment:

A=A _(o) e ^(−qt) , t>T  (10)

[0095] where A_(o) is the initial serum concentration at the moment ofinjection, t=T, and A=0 for 0<t<T.

[0096] Just as the proto-drug entered the individual cells and tissuesof the body according to equation (3), so too will the activation drugenter the individual cells. However, as the drug enters the cells,reactions begin between the proto-drug and the activation drug,affecting the time development of both drugs in the body. Under suchconditions the equations become: $\begin{matrix}{{{C}/{t}} = \left\{ \begin{matrix}{{{gD} - {bC}},} & {0 < t < T} \\{{{gD} - {bC} - {rBC}},} & {t > T}\end{matrix} \right.} & (11) \\{and} & \quad \\{{{B}/{t}} = \left\{ \begin{matrix}{0,} & {0 < t < T} \\{{{fA} - {eB} - {rBC}},} & {t > T}\end{matrix} \right.} & (12)\end{matrix}$

[0097] where r is the reaction rate coefficient, f is the diffusion ratecoefficient for the activation drug into the tissues and cells havingconcentration A. In other words, f for the activation drug correspondsto g for the proto-drug. Similarly, e is the coefficient for B andcorrespondingly b is the coefficient for C.

[0098] Next, the reaction between the proto-drug and the activation drugproduces the toxin in the cells. The concentration of this toxin, J, isgiven by: $\begin{matrix}{{{J}/{t}} = \left\{ \begin{matrix}{0,} & {0 < t < T} \\{{{rBC} - {hJ} - {jJK}},} & {t > T}\end{matrix} \right.} & (13)\end{matrix}$

[0099] where h is the rate coefficient for the loss of the toxin fromthe cell, and jJK gives the reaction rate for the toxin with the vitalcell chemistry at a rate given by j, K being the concentration of thecompound or compounds of the cell chemistry, the loss of which causescell death. The rate at which this vital cell chemical component isdestroyed also depends on both the concentration J of the toxin and onits own concentration K; this rate is given by: $\begin{matrix}{{{K}/{t}} = \left\{ \begin{matrix}{K_{o},} & {0 < t < T} \\{{- {jJK}},} & {t > T}\end{matrix} \right.} & (14)\end{matrix}$

[0100] where K_(o) is the concentration value of K at t<T.

[0101] Equations (2), (10), (11), (12), (13), and (14) can be readilysolved by numerical methods and are significant in that they provide thedetailed time course for the administration of the activation drugrelative to the administration of the proto-drug; furthermore, these sixequations are useful for refining values for R as determined by equation(8) since equation 8 provides the approximate value of R. The solutionof equation (14) also provides a more precise determination of theeffects of toxin exposure that is only approximately given by equations(5), (6), and (7). However, because of their complexities, the use ofthe equations (2), (10), (11), (12), (13), and (14) are somewhatinconvenient to the elucidation of the advantageous means to enhancingthe desired differential toxicity. For example, there is an optimum timedelay period, T. Equation (8) demonstrates that whereas R will rise withthe time delay T, the exposures E_(A) and E_(B) and the correspondingconcentrations C_(A) and C_(B) will drop with T. As a result, timedelays must be chosen that provide sufficient differential toxicitywhile still delivering the needed toxic dose to the targeted tissues.The optimum time T can then be fine-tuned to provide an accuratedetermination using the numerical methods for evaluating equations (2),(10), (11), (12), (13), and (14) already indicated.

[0102] Whereas the optimum time delay can be achieved by means of thecalculations described above, it may be necessary to confirm theadequacy of dosages and of time delays by actual measurements. Inlaboratory animals this can be done directly, as the present inventorshave done. These actual measurements for determining the time delay canbe carried out on the complete proto-drug or on just the compoundcontaining the differentially concentrating moiety, since it is thismoiety that is critical in establishing the concentration differentialbetween normal and cancerous tissue.

[0103] The data determining the optimum time delay of either thecompound containing the differentially concentrating moiety or theproto-drug itself in animal models in vivo can be used to approximatethe time delay in humans by the application of Kleiber's law to theanimal data. Kleiber's law states: “The total metabolic rate for anorganism scales as the body mass raised to the ¾ power.” To determinetime delay, however, the specific metabolic rate, meaning the rate perunit mass is required. The specific metabolic rate is obtained bydividing the above ¾ power Kleiber scaling factor by the ratio of theindividual animal masses for the two species. This results in an overall−¼ power scaling factor for the specific metabolic rate. For example, inthe case of extrapolating mouse model time delay data to humans, acomparison of the mouse body mass (e.g., 20 grams) to the human bodymass (e.g., 80 kilograms) provides a ratio of 4,000. Applying Kleiber'slaw, as adapted for calculating the specific metabolic rate to thisratio, it is estimated that the human specific metabolic rate is 8.0times slower than the mouse. Hence, a 4 day time delay in a mousetranslates to an approximate time delay of 32 days in a human. In thecase of aggressive cancers where a higher risk is required to effect agreater chance of cure rate (i.e., where a greater than usually acceptedtoxicity to normal tissue would be tolerated so as to achieve aneffective toxic level in cancer tissue), time delays shorter than thosepredicted by application of Kleiber's law to the animal model may berequired. Also, for children, the elderly, and patients in very poorhealth, time delays longer than estimated by Kleiber's law may benecessary to ensure minimal toxicity to normal tissue. Time delays froma day to a couple of months are possible.

[0104] Where the time delay period is being used in human or veterinaryclinics, eliminated metabolites of the proto-drug are monitored todetermine when the toxic levels of proto-drug in normal tissues havedropped to safe levels (i.e., to determine when the activation drugshould be administered). That is to say, one can establish beforehandthe maximum dose of the toxin that can be safely tolerated by a patient.Monitoring the excretion of the metabolites using chemical assayprocedures such as HPLC allows determination of the time when theproto-drug has reached safe levels in the normal tissues of thebody—levels such that activation of the proto-drug will result insufficiently low levels of the toxin in the normal tissues of the body.Administration of the activation drug at this time results in themaximum dose of toxin for the targeted cancerous tissues. If the levelof the toxin in the targeted tissues is insufficient, then one mustadminister a higher initial dose of the proto-drug and use a longer timedelay. If such modifications do not provide a sufficiently toxic-dose inthe targeted tissues on activation, then the differentiallyconcentrating moiety, the toxic moiety, or both must be redesigned toprovide superior pharmacokinetic parameters. The examples of theproto-drug and activation drug combinations as tested, however, haveproved to have the necessary superior pharmacokinetic parameters.

[0105] The synergistic effects of pharmaceuticals are a known andfrequently encountered hazard resulting from the administration of twoor more drugs at the same time or in close time proximity to oneanother. By contrast, the present invention requires drug interactionand intends that when administered alone there will be little or noeffect of each of the drugs individually on the body. Moreover, it isintended that the optimum time delays between administration of theproto-drug and the activation drug can be determined and used to avoidharmful side-effects of anticancer, disease fighting drugs.

[0106] The present invention further provides a method of selectivelydelivering a proto-drug and activating such proto-drug to become orrelease a cytotoxic compound in tumor tissue by waiting for a time delayand then administering an activation drug. The present invention furtherprovides a method of selectively delivering the compounds of Formula Iand Formula II and activating the compounds of Formulas I and II tobecome or release a cytotoxic compound in tumor tissue by waiting for atime delay and then administering sodium fluoride.

[0107] The present invention also provides a method of treatingneoplasms in a mammal comprising administering to a mammal in need ofsuch treatment a therapeutically effective amount of a proto-drugcomprising a differentially concentrating moiety, a toxic moiety, and acap moiety, waiting for a time delay and then administering anactivation drug.

[0108] The present invention yet also provides a method of treatingneoplasms in a mammal comprising administering to a mammal in need ofsuch treatment a therapeutically effective amount of a compound ofFormula I or II

[0109] wherein:

[0110] R¹ is SiZ₃;

[0111] R² is methyl, chloroethyl, hydroxyethyl, or bromoethyl;

[0112] R³ is chloroethyl, hydroxyethyl, or bromomethyl;

[0113] R⁴ is H, SO₃H, or taurine;

[0114] R⁵ is SiZ₃;

[0115] R⁶ is H, SO₃H, or taurine;

[0116] each Z of Z₃ is independently t-butyl or methyl;

[0117] X is carbon, oxygen or nitrogen; and

[0118] W is carbon, oxygen, or nitrogen

[0119] waiting for a time delay and then administering sodium fluoride.

[0120] Methods for In Vitro Testing

[0121] In vitro methods used to evaluate compounds and assemblages ofthe present invention rely on principles common to many systemspresently available. For example, monolayer or suspension cells areplated, treated with the proto-drug, and incubated under appropriateconditions for the particular cell type.

[0122] Several parameters of cell health are then calculated orestimated throughout the test period (e.g., 7 days). If the assemblage(i.e., the proto-drug and the activation drug) is to be evaluated by anin vitro method following a time delay period, (e.g., 3 days ofincubation) the activation drug is added to the appropriate wells andthe testing continues for an additional period (e.g., 4 days).

[0123] The concentration of drug that causes cell kill or growth arrestin 50% of the population, IC₅₀, is calculated by a comparison betweenthe drug-treated wells and untreated controls. Another useful parameterthat can be determined by in vitro methods is the activity of the drugversus time. This parameter is of particular importance for those drugsthat are slow acting due to metabolic reactions or poor absorption.

[0124] Murine Tumor Assays

[0125] Inhibition of tumors transplanted into mice is an acceptedprocedure for studying the efficacy of anti-tumor agents. Customarilythe procedure is performed by trocar subcutaneous implantation of piecesof a tumor extracted from a carrier mouse, or by subcutaneous injectionof cancerous cells on the order of 5 million per animal.

EXAMPLES

[0126] The present disclosure presents details of the use of the presentinventive process to design and evaluate an assemblage. Moreover, theinvention includes specific molecule design (e.g. proto-drugs), meansfor the preparation of these compounds, details of their testing, andprocedures for the determination of the time delay. The followingexamples are merely illustrative of the present invention and should notbe considered as limiting the scope of the invention in any way.

Example I

[0127] The preparation of an assemblage as described by the novelprocess set forth above began with the selection of a differentiallyconcentrating moiety. In order to narrow the search for suitablecandidates, the process began with a review of the existing literature.

[0128] Information regarding benzophenoxazines suggested that the use ofsuch compounds tied to a toxin would be effective if the toxic moietycould be activated after the drug had reached a differentialconcentration in the cancer tissue. However, benzophenoxazines otherwisehave poor physicochemical properties (e.g., poor solubility), a problemthat makes them not too attractive as delivery moieties. In addition tothe benzophenoxazine dyes, other substances show tissue selectivity.Among those, the yellow thioxanthones display tissue selectivity andsome anticancer activity (Miller, et al. U.S. Pat. No 5,346,917). Theselectivity of these dyes, coupled with their favorable physicochemicalproperties, makes them good candidates for the selective delivery ofdrugs.

[0129] In Formula I, a yellow dye which is, a thioxanthone, has beenselected for the differential concentration moiety of the proto-drug.Confirmation that a thioxanthone displays differentially concentratingproperties was demonstrated by in vivo examination of normal and tumortissues. The data from this study confirms that yellow thioxanthone hasdifferentially concentrating properties.

[0130] Several mice with implanted palpable tumors (pancreas) wereinjected iv with 40 mg/kg of the thioxanthone yellow dye. Animals weresacrificed at 4 h, 1 day, 2 days, 3 days, 4 days, and 5 days followingadministration of the thioxanthone. The tumor, pancreas, stomach, partof the intestine, and liver were cut and minced in alcohol to extractthe dye. The amount of dye from each tissue was evaluated quantitativelyusing HPLC. A higher concentration of the dye in tumor compared to othertissues initially was seen at 1 day post-administration. There was nosignificant amount of dye remaining in the normal tissue by days 3-4.

[0131] The data from this in vivo study were also used to determine thetime delay for the proto-drug that contains this differentiallyconcentrating moiety. An optimum time delay of 4 days in the mousepredicts a time delay of about 32 days in the human. In the mouse model,however, possible time delays ranged from 2.5 to 6 days, translating toa time delay range of 20 to 48 days in humans. In the case of aggressivecancers, shorter time delays, possibly as short as one day, may benecessary to provide the required level of toxicity in the tumor tissue.By contrast, a time delay of a couple of months may be necessary in theseverely compromised patient.

[0132] The selection of a toxic moiety to be linked to thedifferentially concentrating moiety was carried out by a review of theexisting literature. The powerful anticancer activity of mechlorethaminehas long been attributed to the nitrogen mustard moiety of the molecule.It acts by alkylating biologically important cell constituents whosefunction is then impaired. The major indications for its clinical useinclude bronchogenic carcinoma, Hodgkin's disease, non-Hodgkin'slymphomas, lymphosarcoma, and chronic myelocytic or chronic lymphocyticleukemia. However, nitrogen mustard displays a wider spectrum of actionin vitro than its clinical applications suggest. The limitation on itsuse in vivo is directly related to its toxicity (nausea, vomiting,anorexia, leukopenia thrombocytopenia, local irritation). Attempts havebeen made to improve its selectivity or delivery through itsincorporation into a variety of compounds (for example, Haines, et al.,J. Med. Chem. 30, 542 (1987); Alexander, et al., Tet. Lett. 27, 3269(1991). Thus, mechlorethamine is an excellent candidate for delivery andthe thioxanthone nucleus provides the appropriate vehicle to deliver itto the target tissues and cells. In the example of Formula I, a nitrogenmustard, specifically mechlorethamine, has been selected for thecytotoxic moiety of the proto-drug.

[0133] Another group of powerful anticancer agents is the naturalpodophyllotoxins, which have been used to treat brain tumors and acutegranulocytic leukemias, among others. These substances act by inhibitingmitosis in a reproducing cell. Limitations in their use are similar tothose of nitrogen mustard described above with anemia as an addedtoxicity. Again, these types of substances are good candidates forselective delivery using thioxanthones. In Formula II, a podophyllotoxinderivative has been selected for the cytotoxic moiety of the proto-drug.

[0134] Following the selection of the differentially concentratingmoiety and the toxic moiety, a mask or cap moiety is required tocomplete the elements of the proto-drug. In Formulas I and II, aninorganic moiety, specifically a silicate, SiZ₃, (where each Z of Z₃ isindependently t-butyl or methyl) has been selected for the masking orcapping moiety of the proto-drug. This inorganic silicate moiety, SiZ₃,further has the chemical characteristic that it is highly reactive withfluoride salts, some of which are not significantly toxic. As aconsequence, the moiety is removable by non-toxic or low toxicitychemical reactions that can occur within the patient's body.

[0135] A reaction of this type occurs when fluoride present intoothpastes reacts with the calcium present in teeth. The productformed, a fluoride of calcium, is a very stable and stronger materialthan the natural calcium salt present in teeth. This is an example of adisplacement reaction driven by the formation of a more stable material.In the case of compounds of Formulas I and II, silicon ions have astrong attraction for fluoride, stronger than the attraction of sodiumor potassium for fluoride; thus, a displacement reaction can occur whensodium or potassium fluoride encounters a silicon compound. The reactionis very selective and specific.

[0136] The resulting proto-drug must be activated by an activation drug.In the examples of Formulas I and II, an inorganic chemical, a fluoridesalt, has been selected for the uncapping chemical that will react withthe proto-drug. The specific fluoride salt, sodium fluoride, was chosenbased upon its known chemical properties. This inorganic fluoride saltactivation drug is particularly suited for reaction with the proto-drugsof Formulas I and II because it is highly reactive with the specificsilicates used as the capping moieties in these compounds. Furthermore,sodium fluoride is not significantly toxic in the quantity needed touncap such proto-drugs. As a consequence, the mask or cap moiety of theproto-drug is removable by a very non-toxic inorganic chemical thatoccurs by a reaction within the patient's body without adverse effectson the patient.

Example II

[0137] Given below are the details of the proto-drug of Formula I.

[0138] 1,4-Dihydroxythioxanthone

[0139] A suspension of thiosalicylic acid (5.0 g) and hydroquinone (5.0g) in concentrated sulfuric acid (100 ml) is stirred at room temperaturefor 4h. The red suspension is poured on ice and allowed to reach ambienttemperature when it is then filtered. The solid obtained is treated withsaturated sodium bicarbonate and the solution was carefully neutralizedwith 20% hydrochloric acid. The solid is filtered, dissolved in acetoneand filtered again through a plug of silica gel. The solution isconcentrated and the solid purified by column chromatography with silicagel using a mixture of hexanes:ethyl acetate (1:1) as the eluent. Thefraction containing the product is concentrated under reduced pressureto provide the yellow title compound (2.36 g, 30%).

[0140] FDMS; m/e=244(M⁺)

[0141] Preparation 2

[0142] 1-Chloroethoxy-4-hydroxythioxanthone

[0143] A solution of 120 mg of 1,4-dihydroxythioxanthone in 100 ml ofacetone is stirred for 0.5 h at room temperature with 138 mg ofpotassium carbonate. A solution of 80 mg of 1-bromo-2-chloroethane in 5ml of acetone is then added and the mixture refluxed for 24 h. Theheterogeneous mixture is cooled, filtered, concentrated under reducedpressure and purified by silica gel column chromatography using a 1:1mixture of hexanes and ethyl acetate to produce 100 mg of a yellow-redcrystals (65%).

[0144] FDMS; m/e=307 (M⁺)

[0145] Preparation 3

[0146] 1 -(N,N-bisdiethanolaminoethoxy)-4-hydroxythioxanthone

[0147] A solution of 0.5 g of 1-chloroethoxy-4-hydroxythioxanthone in 5ml of diethanolamine is heated under nitrogen at 110° C. for 1 h. Aftercooling, water is added and the mixture extracted four times with ethylacetate. The ethyl acetate extract is washed with water, dried withsodium sulfate, concentrated and purified by alumina columnchromatography using ethyl acetate:ethanol (4:1) as the eluent solvent.The material obtained is crystallized from acetone to give 0.23 g (38%)of the yellow product.

[0148] FDMS; m/e=376 (M⁺)

[0149] Preparation 4

[0150] 1-(N,N-bischloroethylaminoethoxy)4-hydroxythioxanthone

[0151] Method I: A solution of 0.5 g of1-(N,N-bisdiethanolaminoethoxy),4-hydroxythioxanthone in 5 ml of thionylchloride is heated to reflux for 6 h. The excess. thionyl chloride isremoved by distillation, and the remaining solid is put through analumina column chromatography using ethyl acetate: hexanes (1:1) as theeluent produced 0.3 g (58%) of a yellow solid.

[0152] FDMS; m/e=413 (M⁺)

[0153] Method II: A solution of 0.5 g of1-(N,N-bisdiethanolaminoethoxy)-4-hydroxythioxanthone in 10 ml ofpyridine is treated at low temperature and under nitrogen with 0.4 g ofmethanosulphonyl chloride and the mixture kept under refrigeration for24 h. The mixture is then poured in water and extracted with ethylacetate. The ethyl acetate extract is washed with water, dried oversodium sulfate and concentrated to a yellow solid. This solid isdissolved in dimethylformamide (5 ml) and stirred under nitrogen at 80°C. with 3 g of lithium chloride for 24 h. The mixture is cooled, mixedwith water and extracted with ethyl acetate. The ethyl acetate extractis washed with water, dried with sodium sulfate and concentrated.Purification via alumina chromatography using ethyl acetate:hexanes(1:1) as the eluent produces 0.37 g (71%) of the yellow solid.

[0154] FDMS; m/e=413 (M⁺)

[0155] Preparation 5

[0156]1-(N,N-bischloroethylaminoethoxy)-4-tert-butyldimethylsilyloxythioxanthone

[0157] A solution of 0.50 g of1-(N,N-bischloroethylaminoethoxy)-4-hydroxythioxanthone in 10 ml ofdimethylformamide is mixed with 0.22 g of tert-butyldimethylsilylchloride, 0.20 g of imidazole and a catalytic amount ofdimethylaminopyridine, and stirred at room temperature for 12 h. Thesolution is then added over water and extracted with ethyl acetate. Theethyl acetate extract is washed sequentially with saturated sodiumbicarbonate and water, dried over sodium sulfate, concentrated anddissolved in a mixture of ethyl acetate:hexanes (1:4) and passed througha plug of alumina. The solid obtained after concentration is pure andweighed 0.48 g (70%).

[0158] FDMS; m/e=527(M⁺)

Example III

[0159] 1-(N-chloroethyl-N-methylaminoethoxy)-4-hydroxythioxanthone

[0160] A solution of 0.5 g of 1,4-dihydroxythioxanthone in 20 ml ofdimethylformamide is stirred for 0.5 h at room temperature with 6 g ofpotassium carbonate underanhydrous conditions. Mechlorethaminehydrochloride (0.4 g) is then added and the heterogeneous mixture wasstirred for 12 h at 50° C. Water is added and the mixture extracted withethyl acetate. The ethyl acetate solution is washed with water, driedwith sodium sulfate, passed through a plug of alumina and concentratedunder vacuum to produce 0.4 g (55%).

[0161] FDMS; m/e=364 (M⁺)

Example IV

[0162] Compounds of Formula I were tested in vitro using murine lines ofleukemia L1210 cells, pancreas tumor (Pan 03), Lewis lung carcinoma, anda normal fibroblast. The IC₅₀ value for all the assemblages, meaning aproto-drug of Formula I followed by the activation drug sodium fluoride,were lower than 0.4 micromolar for the cancerous tissues. Comparablevalues were obtained for the normal fibroblast cells in vitro. In thecase of testing of the complete proto-drug without subsequent treatmentwith an activation drug, the IC₅₀ was 0.1 molar in normal cells. Thetest demonstrated that activation significantly altered the toxicity ofthe proto-drug.

Example V

[0163] A study of the activity of compounds of Formula I has beenperformed on mice using three tumor models: leukemia, lung and pancreas.A total of 5 animals per tumor line were used and treatment wasinitiated two weeks after implantation (late stage testing). Theproto-drug compound was administered three times at a dose of about 30%that of the mouse LD₅₀ of the mechlorethamine toxic moiety, followed bythe activation drug, sodium fluoride, dosed (in excess) at 5 times themolar equivalent, 4 days later.

[0164] Experiment: C57 male mice (5/tumor line) were implantedsubcutaneously with ca. 1 million cancerous cells suspended in salinesolution, and tumors were allowed to grow for 2 weeks when they werepalpable. Test compound, 0.2 ml, was then administered ip as asuspension containing water, polyethylene glycol (3%) and alcohol (5%).The activator was administered 4 days later as an ip solution. Two moretreatments were given with a week intermission between them.

[0165] Results: Lung tumors were unaffected by the treatment and animalswere sacrificed before the end of the 30-day study. Treatment ofleukemia infected animals produced 4 cures (out of 5 animals) asevidenced by disappearance of tumor; three of five animals implantedwith pancreas tumor cells were cured following the drug treatmentregimen. Cured animals were maintained beyond the 30-day study term.Other animals were sacrificed.

[0166] A second set of experiments produced 5/5 cures in both solidleukemia L1210 and pancreatic tumors.

1. An assemblage comprising a substantially biologically inertproto-drug and a substantially biologically inert activation drug,whereby the proto-drug comprises a differentially selective moiety, atoxic moiety and a cap moiety and whereas the moieties of the proto-drugare linked together in such a manner as to make the proto-drug itselfsubstantially inert.
 2. A process for the preparation of a substantiallybiologically inert proto-drug whereby the process comprises: (a)selection of a differentially concentrating moiety by a method chosenfrom the group consisting of differential HPLC, differentialchromatography, and in vivo differential rate analysis; (b) selection ofa toxic moiety by a method chosen from the group consisting of in vitrotesting, in vivo testing and evaluation of published lists of toxicmoieties; (c) selection of a cap moiety by a method chosen from thegroup consisting of in vitro testing, in vivo testing and evaluation ofpublished lists of reagents with the toxic moiety; and (d) linking thedifferentially concentrating moiety, the toxic moiety, and the capmoiety in such a manner as to make the proto-drug itself substantiallybiologically inert.
 3. A process for the preparation of an assemblage,whereby the process comprises: (a) selection of a differentiallyconcentrating moiety by a method chosen from the group consisting ofdifferential HPLC, differential chromatography, and in vivo differentialrate analysis; (b) selection of a toxic moiety by a method chosen fromthe group consisting of in vitro testing, in vivo testing and evaluationof published lists of toxic moieties; (c) selection of a cap moiety by amethod chosen from the group consisting of in vitro testing, in vivotesting and evaluation of published lists of reagents with the toxicmoiety; (d) selection of an activation drug by a method chosen from thegroup consisting of in vitro testing, in vivo testing and evaluation ofpublished lists of reagents with the cap moiety; and (e) linking thedifferentially concentrating moiety, the toxic moiety, and the capmoiety in such a manner as to make the proto-drug itself substantiallybiologically inert.
 4. A method of treating neoplasms in a mammal, suchmethod comprising: (a) administering to a mammal in need of suchtreatment an effective amount of a proto-drug, such proto-drugcomprising a differentially concentrating moiety, a toxic moiety and acap moiety; (b) waiting for a time delay period; and (c) administeringto the mammal an activating amount of an activation drug whereby theactivation drug converts the proto-drug in vivo to a pharmacologicallyactive compound.
 5. A method of converting a substantially biologicallyinert compound to a pharmacologically active agent, such methodcomprising: (a) administering to a mammal a proto-drug, such proto-drugcomprising a differentially concentrating moiety, a toxic moiety, and acap moiety whereby the moieties are linked together in such a fashion asto create a biologically inert compound; (b) waiting for a time delayperiod; and (c) administering to the mammal an activation amount of anactivation drug whereby the activation drug converts the proto-drug to apharmacologically active agent.
 6. A method of selectively delivering acytotoxic compound to tumor tissue, such method comprising administeringto a mammal a proto-drug comprising a differentially concentratingmoiety, a toxic moiety and a cap moiety, whereby the proto-drug deliversa cytotoxic compound to the tumor tissue in such a manner as to preventsignificant damage to normal tissues by maintaining the cap moiety onthe proto-drug until the proto-drug differentially concentrates in thetumor tissue during a time delay, and after such time delay theproto-drug produces a cytotoxic compound upon administration of anactivation drug.
 7. A pharmaceutical preparation comprising: (a) aneffective amount of a proto-drug together with a pharmaceuticallyacceptable excipient; and (b) an activating amount of an activation drugtogether with a pharmaceutically acceptable excipient whereby theproto-drug and the activation drug are packaged for individualadministration. 8-24. (Cancelled)
 25. A method of determining a timedelay period between administration of a proto-drug and an activationdrug which comprises determining time T in the equation R=E _(A) /E_(B)=(b _(B) /b _(A)) exp[(b _(B) −b _(A))T] whereby: R is the ratio ofthe diffusion constants of cell types A and B; E_(A) is the exposure ofcell type A to the proto-drug; E_(B) is the exposure of cell type B tothe proto-drug; b_(A) is the elimination constant of cell type A; andb_(B) is the elimination constant of cell type B.
 26. The method ofclaim 25 whereby the time delay period is evaluated by in vivoprocedures.
 27. A proto-drug comprising: (a) a thioxanthone moiety thatacts as a differentially concentrating moiety; (b) a mechlorethamine orpodophyllotoxin moiety that acts as a toxic moiety; and (c) a silanemoiety that acts as a cap moiety whereby the thioxanthone,mechlorethamine or podophyllotoxin, and silane moieties are linked toform a substantially biologically inert compound. 28-33. (Cancelled).34. An assemblage comprising a substantially biologically inertproto-drug and a substantially biologically inert activation drug,whereby the proto-drug comprises (a) a thioxanthone moiety that acts asa differentially concentrating moiety; (b) a mechlorethamine orpodophyllotoxin moiety that acts as a toxic moiety; (c) a silane moietythat acts as a cap moiety; and the activation drug is a fluoride salt.35. The assemblage of claim 34 whereby the fluoride salt is sodiumfluoride.